Altered pore properties and kinetic changes in mutants of the Vibrio cholerae porin OmpU.
The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and beta-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.
Lauman
Brian
B
Department of Biology & Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Pagel
Melissa
M
Delcour
Anne H
AH
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000494:pharmacology*; D003840:Deoxycholic Acid / Q000494:pharmacology; D004594:Electrophysiology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D010539:Permeability / Q000187:drug effects; D011134:Polysaccharides / Q000494:pharmacology; D016062:Porosity; D014734:Vibrio cholerae / Q000737:chemistry* / Q000472:pathogenicity; D018440:beta-Lactam Resistance
D018829:Adhesins, Bacterial; C096349:OmpU protein, Vibrio cholerae; D011134:Polysaccharides; D003840:Deoxycholic Acid; C008315:maltodextrin